HLA allele associations in idiopathic recurrent spontaneous abortion patients from India

BACKGROUND: Rejection of semiallogenic foetus in recurrent spontaneous abortion (RSA) has been postulated to be a consequence of genetic and immunological phenomena. AIM: To evaluate the role of human leukocyte antigen (HLA) alleles in RSA in Indian couples. SETTINGS AND DESIGN: A case-control study. MATERIALS AND METHODS: Eighty-one randomly selected couples with unexplained three or more RSAs and a control group of 97 couples with live birth belonging to the same ethnic background, referred to the Gynaecology Department, KEM Hospital were included in the case-control study. Serological HLA A and B typing was done followed by molecular subtypes, defined using PCR-SSOP technique for HLA A, B, and C in 40 couples and DRB1* and DQB1* in 28 couples which were then compared with appropriate case 46 and 88 controls. RESULTS: Serologically A3 (15.43% vs . 4.43%; odds ratio (OR) = 4.34; P = 0.0002) and B17 (25.3% vs . 11.34%; OR = 3.49; P = 0.0001) were increased. Haplotype A1-B17 was significantly increased. Molecular subtyping revealed that A*030102 (11.25% vs . 4.34%; OR = 3.00; P = 0.07), B*5701 (11.25% vs . 1.08%; OR = 13.10; P = 0.003), Cw*120201 (25% vs . 4.34%; OR = 10.50; P = 2.05E-05), HLA DRB1*030101 (17.85% vs . 3.40%; OR = 7.6; P = 0.0001), DRB1*150101 (32.14% vs . 13.63%; OR = 4.8; P = 0.0003), and DQB1*060101 (35.71% vs . 29.34%; OR = 2.3; P = 0.004) were significantly increased in patients. A differential association was noticed when compared with reported world RSA patients. CONCLUSION: The HLA alleles A*030101, B*5701, Cw*120201, DRB1*030101, and DRB1*150101 as well as their associated ancestral haplotype may play a significant role in development of RSA in India.

Recurrent spontaneous abortion (RSA) is deÞ ned as a sequence of three or more consecutive spontaneous abortion. RSA is a heterogeneous condition, which may have many possible causes; more than one contributory factor is suggested to underlie the recurrent pregnancy losses. The major causes are att ributed to genetic, endocrinological, or immunological. In the immunological cause, it may be autoimmunity or alloimmunity. In autoimmune, a woman may develop antiphospholipid antibodies, antithyroid antibodies, antinuclear antibodies, and antineutrophilic cytoplasmic antibodies. Alloimmune cases of RSA show involvement of human leukocyte antigen (HLA) and blood group antigens. The literature review reveals association of HLA alleles with RSA in various populations. [1][2][3][4][5][6][7][8][9][10][11][12][13] Strong association of HLA DR alleles have been shown with RSA. [4][5][6][7][8][9] Numbers of studies indicate that there is a high sharing of HLA antigens in couples with RSA. In 1977, Komlos and his associates demonstrated the HLA allele sharing hypothesis concerning RSA. [14] Several studies have confirmed HLA antigens sharing between spouses in RSA. [12][13]15] Further meta-analysis of selected case-control studies suggested a slightly increased and significant risk of RSA among couples who shared at least one allele in HLA DR locus. [15] Other studies have also found nonsharing of HLA antigens. [16,17] In India, few studies have been conducted on HLA sharing, antipaternal cytotoixic antibodies, anticardiolipin antibodies, and lupus anticoagulant in RSA patients. [18,19] Studies from 120 Indian RSA women for nonclassical HLA G and HLA E associations have revealed that E*0101 and G*010103 were signiÞ cantly associated for the Þ rst time. [20,21] Further, the role of 14 bp deletion in the HLA G gene toward the maintenance of pregnancy has shown no frequency diff erence among controls and RSA patients. [22] Unexplained RSAs has many etiologies, many of them have immune dysfunctions which Shankarkumar

MATERIALS AND METHODS
A total of 81 couples with three or more RSAs and control group of 97 couples who had one or more children referred to the Gynaecology Department of KEM Hospital were included in the study. The RSA patients and the controls were in their reproductive age varying from 20 to 40 years. All the 81 couples were negative for conventional causes like hypertension, diabetes, infections due to toxoplasma, rubella, etc., hormonal dysfunction, thrombophilic risk factors, cytogenetics, autoimmune, and anatomical abnormalities. HLA serological typing and anti-HLA antibodies were done following the standard two-stage NIH microlymphocytotoxicity assay. In short from each individual peripheral blood was collected in heparin anticoagulant and the lymphocytes were separated on histopaque by density gradient centrifugation and the HLA A and B alleles were identiÞ ed using speciÞ c HLA antisera of commercial and indigenous origin. Each specificity was defined by using three sera with good coefficient of correlation "r" value. The anti-HLA antibodies were investigated against the husband's lymphocytes. Further, molecular characterization of the HLA alleles were done by PCR-SSOP technique in 40 patients for HLA A, B, and C loci, in 28 patients for DRB1 and DQB1 loci which were then compared with appropriate 46 and 88 controls. The HLA antigen association between patients and normal controls were evaluated for allele frequency estimate, haplotype analysis, etiologic fraction, preventive fraction χ 2 -test, P-value, and odds ratio as described earlier. [23]

RESULTS
Our analysis revealed that HLA A1, A3, and B17 were signiÞ cantly increased while A9 decreased among the RSA patients from western India [ Table 1]. Among the results of the 40 RSA patients characterized for their molecular A, B, and Cw subtypes compared with the normal controls.
It was interesting to note that A*030101, B*5701, and Cw*120201 subtypes were signiÞ cantly increased [ Table 2]. Further, HLA DRB1 and DQB1 molecular typing among 28 RSA patients revealed that DRB1*030101, DRB1*150101, and DQB1*060101 were significantly increased while DRB1*010101, DRB1*110101, and DRB1*100101 were decreased among the RSA patients [ Table 3]. Our comparative analysis from diff erent world RSA patients revealed that our patients had diff erential association [ Table 4]. Haplotype sharing has been signiÞ cantly observed among 72 out of the 81 RSA couples (88.00%). Two locus haplotype analyses reveal that haplotype A1-B17 is signiÞ cantly increased as well as haplotypes A10-B8, A3-B7, and A9-B35 were observed among RSA couples [ Table 5]. The anti-HLA antibodies were found to be positive in 14.7% of the RSA women.

DISCUSSION
The signiÞ cant observations of the present study are (1) HLA A comparative analysis of HLA in RSA patients reported from the world revealed a diff erential association among our patients. Earlier we had reported HLA A3 association in RhD immunised women from western India. [24] HLA B17 has been reported to be associated in Italian RSA patients. [11] HLA B17 has also been implicated in South Indian psoriasis patients. [25] Recently, haplotype A1-B17 has been reported to be involved in antineutrophil cytoplasmic antibodies (ANCA) production from western Indian population. [26] A signiÞ cant reduced frequency of HLA B35 has been described in Japanese RSA patients. [12] Among Roman RSA couples, signiÞ cant diff erences in HLA A2, B18, and B40 have been reported. [13] Increased frequencies of Cw5, Cw6, and DR2 in Italian RSA patients have been reported. [1] Further among the UK RSA patient population linkage disequilibrium between A2 and B12 has been reported. [2] Various studies on HLA DR and DP loci have shown associations with RSA in Italian, [10] Danish, [6,9] and Japanese [5,7,8] populations. Recently, HLA DRB1*1502 has been reported to be signiÞ cantly associated in Japanese RSA patients. [4] The reports on antigen sharing in couples with recurrent miscarriage led to several studies assessing the role of HLA antigens and their inß uence on the outcome of pregnancy. [27] In fact, couples sharing at HLA A, HLA B, HLA C, HLA DR, and HLA DQ loci has been reported to be positively associated with the risk of  [14] The discrepant results in the HLA and RSA studies were att ributed to inconsistent deÞ nitions of recurrent miscarriage and of the control groups, the small number of samples, and the diff erent tissue typing methods used to investigate the HLA loci. Therefore, we had done serology initially and then molecular characterization for the allele subtypes in this case-control study to deÞ ne all the HLA loci.
Due to variation in immunization and the resultant RSA. [28] Since 1977, increased HLA sharing among spouses has been associated with RSAs; later more speciÞ c HLA DR and/or DQ antigens were suggested. HLA sharing has also been reported in couples that fail to achieve pregnancy with multiple cycles of assisted reproductive techniques. Studies examining the association of HLA sharing with   outcome, a wide variation in the incidence of anti-HLA antibodies in the sera of pregnant women has been reported in the literature. The values ranged from 7.3 to 36%. [24] It has been reported earlier that 27.8% of RSA women had anti-HLA antibodies from northern India. [18] In our study, we have found an incidence of 14.7% anti-HLA antibodies, which suggests that the blocking factors as well as immunity toward the husband's lymphocytes could be different among western Indian RSA couples.
Recently, the involvement of killer cell immunoglobulin-like receptors (KIR) in abortion with speciÞ c HLA molecules on trophoblast (HLA C, G, and E) may inß uence the outcome of pregnancy has been suggested. [29] Studies conducted in reproductive immunology component of the 14th International Histocompatibility Workshop has suggested that women with alloimmune abortions or implantation failures may possess a rather uncommon KIR repertoire characterized by increased presence of activating receptors and may have a higher potential decidual natural killer (NK) cell activation, thus increased risk for an adverse reproductive outcome. [30] It could be possible that HLA Cw*12021, the associated allele along with its associated ancestral haplotypes A3-B17-CwBk-DR3 in India could have an immunopathological role via the HLA C-KIR related activation which leads to an adverse reproductive outcome. This remains to be still elucidated though we have proved that ancestral haplotype A1-B17-Cw6-DR7 has been associated with ANCA production in renal-associated vasculitis from India. Further we have provided evidence of Cw*1507 allele to be strongly associated in HIV-1 infected Indian patients (personnel communication). Future studies involving these parameters along with the other immunological evaluations are necessary to substantiate the immunological basis of rejection in RSA.